The overall objective of this research proposal is to understand the biogenesis of the (Na ion plus K ion)-activated adenosine triphosphatase (Na,K-ATPase). We have chosen two model systems for study: the electric organ of the eel, Electrophorus electricus, and the embryonic tissue of the brine shrimp, Artemia salina. These systems are rich in the Na,K-ATPase, and methods for purification of the enzyme have been developed. The electric eel Na,K-ATPase subunits have been labeled with radioactive valine and isolated. The synthesis rate will be calculated after the specific radioactivity of the valyl t-RNA has been measured. Since a lag period between incorporation and isolation of labeled Na,K-ATPase subunits has been observed, further studies will determine if this lag period represents transit time. Protein synthesis inhibitors will be tested to see whether the lag period can be altered. Newly syntesized Na,K-ATPase subunits will be isolated by immunoprecipitation or by density modification with 2H, 13C, 15N amino acids. Biochemical analysis of the eel subunits in transit will reveal whether they are membrane-bound and if the subunits are transported before or after assembly. Immunocytochemical localization of the eel subunits during transient will reveal the subcellular structures involved in their transport. The brine shrimp Na,K-ATPase can be labeled with NaH14CO3 during embryogenesis. The lag period between synthesis of the subunits and assembly of the holoenzyme, and the specific rate of synthesis of each subunit will be studied by pulse-chase experiments with NAH14CO3. The site of synthesis of the Na,K-ATPase subunits will be examined in isolated membrane-bound and free polyribosomes by methods of iodinated antibody binding and by cell-free translation of immunoprecipitated mRNA. The route of transport of the subunits to the plasma membrane will be determined by subcellular fractionation and detection by iodinated antibody binding and by immunoelectrophoresis. Isozymes of the brine shrimp Na,K-ATPase have been found, and these will be further tested by pulse-chase experiments with NAH14CO3 and by peptide mapping to determine if a precursor-product relationship exists.